Regulation of intestinal calcium transport by 1,25D occurs at multiple steps and by multiple mechanisms. This proposal focuses on the events between entry of calcium across the brush border membrane and accumulation of calcium in vesicles forming in the terminal web which transport calcium through the cell to the basolateral membrane. The hypothesis is that the 119 kDa calmodulin binding protein, brush border myosin 1 (BBM1), linking the plasma membrane to the actin core of the microvillus, is responsible for the transport of calcium out of the microvillus into these vesicles. The working model is that calcium entering the microvillus binds to calmodulin which then activates BBM1 to move down the actin core to the terminal web where it unloads the calcium to the calbindin containing vesicles for transport through the cytoplasm. Dr. Bikle postulates that 1,25D regulates this function by altering the binding of BBM1 to calmodulin and the membrane by post-translational modifications, and by inducing a tropomyosin-like protein called zipper protein (ZP) which governs the binding of actin to BBM1 and activation of its ATPase. Dr. Bikle's laboratory will test this hypothesis first by examining the pathway through which calcium moves during 1,25D-stimulated calcium transport in Caco2 cells, a human colon cancer cell line that differentiates spontaneously in vitro into an intact epithelium capable of 1,25D-regulated calcium transport. This will be performed using ion capture cytochemistry, proton induced x-ray emission, and fluorescence cell imaging to localize and quantitate the movement of calcium in the cell following 1,25D administration. It will then be determined whether the calmodulin/BBM1 complex and zipper protein are required for calcium transport in Caco2 cells. This will be accomplished using calmodulin inhibitors, antisense constructs to block BBM1 and zipper protein production, mutants of BBM1 and zipper protein lacking critical domains to function as dominant negatives, and zipper protein overexpression to block BBM1 interaction with actin. These manipulations will all be tested for their ability to block 1,25D-stimulated calcium transport.